1. Field of the Invention
This invention is related to the field of synthetic polymers. More specifically, this invention relates to methods, kits and compositions suitable for modulating the solubility of synthetic polymers and, in particular, peptide nucleic acid (PNA) oligomers.
2. Description of the Related Art
Peptides and nucleic acids are naturally occurring compositions which are increasingly utilized in research, diagnostic and therapeutic applications. Though naturally occurring peptides and nucleic acids are generally soluble in aqueous solutions, the solubility of individual compositions of differing sequence can vary substantially, with certain compositions exhibiting little or no solubility in aqueous solution. Additionally, the introduction of products and methods for the synthetic production of peptides and nucleic acids has made available sequence variations which are not known to, or may in fact not, exist in nature. The absence of certain biopolymer sequences in nature may at least partially be due to the limited solubility of the composition.
The limited solubility of certain peptide and nucleic acid oligomers can prohibit what would otherwise be a useful research, diagnostic or therapeutic application for that polymer. Therefore, methods and compositions suitable for improving the solubility of peptides and nucleic acids in aqueous solutions may prove essential to the enablement of new technology which utilizes peptides and nucleic acids which otherwise have little intrinsic water solubility. However, compositions which modulate the solubility of synthetic polymers should preferably be simple and achiral since the effectiveness of complex macromolecules such as nucleic acids and peptides in research, diagnostic or therapeutic applications can be adversely affected by the size, complexity or chirality of attached ligands.
Peptide nucleic acids (PNAs) are non-naturally occurring polyamides (also properly characterized as pseudopeptides) which can hybridize to nucleic acids (DNA and RNA) with sequence specificity. (See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or 5,786,571 and Egholm et al., Nature 365: 566-568 (1993)). PNAs are candidates for investigation as alternatives/substitutes to nucleic acid probes in probe-based hybridization assays because they exhibit several desirable properties. In preferred embodiments, PNAs are achiral polymers which hybridize to nucleic acids to form hybrids which are more thermodynamically stable than a corresponding nucleic acid/nucleic acid complex (See: Egholm et al., Nature 365: 566-568 (1993)). Being non-naturally occurring molecules, they are not known to be substrates for the enzymes which are known to degrade peptides or nucleic acids. Therefore, PNAs should be stable in biological samples, as well as, have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength under conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid. (See: Egholm et. al., Nature, p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (See: Tomac et al., J. Am. Chem. Soc. 118: 5544-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (See. Egholm et al., Nature, p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay appears to be somewhat sequence dependent (See: Nielsen et al. Anti-Cancer Drug Design 8: 53-65 (1993) and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)). As an additional advantage, PNAs hybridize to nucleic add in both a parallel and antiparallel orientation, though the antiparallel orientation is preferred (See: Egholm et al., Nature, p. 566).
PNAs are synthesized by adaptation of standard peptide synthesis procedures in a format which is now commercially available. (For a general review of the preparation of PNA monomers and oligomers please see: Dueholm et al., New J. Chem. 21: 19-31 (1997) or Hyrup et. al., Bioorganic and Med. Chem. 4: 5-23 (1996)). Labeled and unlabeled PNA oligomers can be purchased (See: PerSeptive Biosystems Promotional Literature: BioConcepts, Publication No. NL612, Practical PNA, Review and Practical PNA, Vol. 1, Iss. 2) or prepared using the commercially available products.
Limited aqueous solubility and a tendency toward self-aggregation has been a long established and well documented restriction on applications of PNA (See for example: Lee, Morse and Olsvik, Nucleic Acid Amplification Technologies: Application to Disease Diagnositics, Chapter 3 by Ørum et al., BioTechniques Book Div. of Eaton Publishing (1997) pp. 29-48, at p. 40, Ins. 14-26; Corey, D. R., TIBTECH, 15: 224-229, June (1997) at p. 225, col. 1, In. 37 to col. 2, In. 2; p. 226, col. 2, Ins. 24-30 and p. 229, col. 1, Ins. 14-33; Lesnik et al., Nucleosides and Nucleotides, 16: 1775-1779 (1997) at p. 1775, Ins 1-5; Peyman et al., Angrew. Chem. Int. Ed. Engl., 35: 2636-2638 (1996) at p. 2636, col. 1, Ins. 13-24; van der Laan et al., Tetrahedron Letters, 37: 7857-7860 (1996) at p. 7857, Ins. 1-10; Bergman et al., Tetrahedron Letters, 36: 6823-6826 (1995) and Egholm et al., J. Am. Chem. Soc., 114: 1895-1897 (1992). The solubility properties of PNA oligomers in aqueous solution is known to be very sequence dependent. Purine-rich PNA oligomers are known to be particularly difficult to purify and/or characterize at least partially due to their limited solubility. Similarly, the solubility of PNA tends to decrease as the polymer length increases thereby resulting in a preference for shorter PNAs. Self-aggregation is another property which tends to limit the utility of PNA oligomers. Because certain PNA oligomers cannot be adequately purified or characterized, there, are presently a large number of potentially useful PNA sequence variations which are unavailable for evaluation in research, diagnostic or therapeutic applications.
By way of example, the product literature of a commercial vendor of custom PNA oligomers states xe2x80x9cFor most applications an oligomer of 12-15 is optimal. Longer PNA oligomers, depending on the sequence, tend to aggregate and are difficult to purify and characterizexe2x80x9d (See: Guidelines For Sequence Design of PNA Oligomers: PerSeptive Biosystems, Inc. Promotional Literature; 1997-1998 Synthesis Products Catalog, col. 2, Ins. 6-11, p. 45). Additionally, this document sets forth several rules for the design of a PNA oligomer which will avoid these limitations. Under the heading xe2x80x9cSpecific Design Rulesxe2x80x9d (col. 3, Ins. 1-18), the text reads xe2x80x9cLength: We will not synthesize any sequences with more than 18 bases, not including linkers, amino adds and labels. Purine Content: Purine rich PNA oligomers tend to aggregate and have low solubility. To avoid that follow these specific guidelines: 1. Of any stretch of 10 bases in the sequence do not have more than 6 purines 2. No more than 4-5 purines in a row, specifically no more than 3 G""s in a rowxe2x80x9d. The vendor suggests that one consider analyzing the other strand if it is otherwise impossible to comply with the limitations set forth in guidelines 1 and 2.
A number of modifications have been made to peptide nucleic acids (PNAs) in order to improve their aqueous solubility or minimize polymer self-aggregation. A commonly used modification of PNA which was first used by the inventors involves the incorporation of one or more positively charged terminal lysine residues (See: Egholm et al., J. Am. Chem. Soc., 114: 1895-1897 (1992) at p. 1896, col. 1, In. 23 to col. 2, In. 2). The inventors of PNA, as well as others, have also advocated the preparation of PNAs having backbone modifications which comprise one or more alkyl amine groups (See: U.S. Pat. No. 5,719,262 and Lesnik et al., Nucleosides and Nucleotides, 16: 1775-1779 (1997)). Though these modifications improve aqueous solubility, they also introduce chiral atoms to which are linked moieties having nucleophilic primary amine groups which are positively charged at physiological pH. The introduction of chiral centers into PNA can alter the hybridization properties of the polymer (See: Lee, Morse and Olsvik, Nucleic Acid Amplification Technologies: Application to Disease Diagnositics, Chapter 3 by Ørum et al., BioTechniques Book Div. of Eaton Publishing (1997) pp. 29-48, at p. 33, In. 4, to p. 34, In. 12). Additional nucleophilic moieties and particularly primary and secondary amino groups must be protected during synthesis and their presence can complicate synthesis, labeling and/or purification. Though positively charged PNAs may exhibit improved hybridization kinetics (See: Corey et al., J. Am. Chem. Soc., 117: 9373-9374 (1995) and Corey et al., FASEB Journal, 9, A1391 (1995)), positively charged groups may also result in non-nucleobase specific interactions which may lead to increased background in a hybridization-based assays.
Another approach to overcoming the limited solubility and self-aggregation of PNA has been to modify the backbone to incorporate negatively charged phosphate moieties (See: Peyman et al., Angew. Chem. Int. Ed. Engl., 35: 2636-2638 (1996) and van der Laan et al., Tetrahedron Letters, 37: 7857-7860 (1996)). However, one of the most advantageous properties of PNA is the neutral backbone which allows for nucleic acid hybridization which is fairly independent of ionic strength and is favored at low ionic strength under conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid. Backbone modifications which re-introduce a negative charge will likely negate this advantageous property.
Still another approach to overcoming the limited solubility and self-aggregation of PNA has been to prepare PNA-DNA chimeras wherein the negative charge on the DNA part of the chimera reduces the tendency toward self-aggregation and thus improves solubility (See: Uhlmann et al., Angew. Chem. Ed. Engl., 35: 2632-2635 (1996) at p. 2632, col. 2, Ins. 33-35). However, PNA-DNA chimeras are segmented molecules which exhibit hybrid properties For example, the Tm of chimeras examined by Uhlmann et al. were approximately half way between the Tm of the DNA/DNA hybrid and the PNA/DNA hybrid (See: Uhlmann et al., Angew. Chem. Ed. Engl., 35: 2632-2635 (1996) at FIG. 4).
Though not expressly designed or sold to improve PNA solubility, applicants have noted that a commonly used etherbased, achiral hydrophilic straight chain linker (8-amino-3,6-dioxaoctanoic acid) can be used to minimally improve the aqueous solubility of PNA oligomers and particularly PNAs labeled with hydrophobic moieties such as fluorescein and rhodamine dyes. However, the 8-amino-3,6-dioxaoctanoic acid linker moiety is not branched, does not maintain the proper spacing for nucleobase to nucleobase interactions, does not branch from the backbone (typically made part of the backbone) and furthermore, conveys only a very limited improvement in aqueous solubility to the PNA oligomer.
Because the utility of a particular PNA oligomer in a research, diagnostic or therapeutic application will generally be specifically related to its sequence, the above mentioned limitations on sequence diversity may prove to be an Achilles Heel of this newly developed and very promising technology. Therefore, it would be useful to provide methods, kits and compositions suitable for improving the aqueous solubility of PNA oligomers and/or reducing their tendency toward self-aggregation so that a greater number of pure PNA oligomers are available for use in research, diagnostic and therapeutic applications. The preferred methods, kits and compositions will exhibit little or no adverse effects on the hybridization properties or physical characteristics of the PNA oligomer. Thus, the most preferred solubility enhancing modifying moieties will be achiral, non-nucleophilic and uncharged at physiological pH or achiral, non-nucleophilic and positively charged at physiological pH.
Any methods, kits and compositions which enhance the solubility of PNA oligomers, should also be equally useful in improving the solubility of peptides or polyamide and/or reducing or eliminating self-aggregation of the polymer. With certain variations, similar compositions should find utility for the modification of nucleic acids and nucleic acid analogs.
As previously discussed, the limited solubility of PNA oligomers, and particularly purine-rich oligomers, is well documented in the chemical literature. Though not exclusively a problem associated with purine-rich sequences, it has been observed that the solubility/self-aggregation properties of PNA oligomers are a sequence specific phenomenon which tends to be particularly problematic when preparing purine-rich, and particularly G-rich PNAs. Given these limitations which were encountered when attempting to synthesize, purify and characterize certain desired PNA oligomers, applicants were compelled to invent a means to overcome the limitations of the prior art to thereby obtain the purified PNAs of desired nucleobase sequence which they required. Guided by the discussion contained herein it will become apparent to those of skill in the art that the compositions developed by applicants, or modifications thereof, will find utility in improving the solubility of synthetic polymers such as peptides, other polyamides, nucleic acids, nucleic acid analogs and particularly the nucleic acid analogs which comprise a neutral backbone.
In one embodiment, this invention pertains to branched or multiply branched compositions useful for improving the solubility of synthetic polymers and/or which minimize or eliminate polymer self-aggregation. These branched or multiply branched solubility enhancing compositions may, depending of the nature of the starting materials, generate soluble polymers having modifying moieties which are positively charged or uncharged (at physiological pH), nucleophilic or non-nucleophilic and chiral or achiral, though they are preferably achiral. A preferred combination of the aforementioned variables is a branched or multiply branched modifying moiety which is uncharged, non-nucleophilic and achiral.
Another preferred combination of the aforementioned variables is a branched or multiply branched modifying moiety which is positively charged, non-nucleophilic and achiral. Certain compositions of this invention are particularly well suited for modifying synthetic nucleic acids and its synthetic analogs while other compositions are better suited for the modification of peptides, PNAs and other polyamides.
For the modification of synthetic nucleic acids and its analogs, this invention provides several branched or multiply branched compositions or polymer synthons which are suitable for use in standard nucleic acid assembly methodologies. Preferred compositions shall be phosphoramidite derivatives and preferably, xcex2-cyanoethylphosphoramidites. One very useful xcex2-cyanoethylphosphoramidite is a multiply branched synthon having the formula: 
Because they enhance the solubility of nucleic acids, these compositions are particularly well suited for improving the solubility of nucleic acid analogs in which the sugar phosphate backbone has been modified so that the analog backbone is uncharged.
In another embodiment, this invention also relates to synthetic nucleic acid and nucleic acid analogs which are modified with simple, branched or multiply branched compositions to thereby improve polymer solubility and/or minimize or eliminate polymer self-aggregation.
For the modification of polyamides, peptide and PNAs, this invention provides several branched or multiply branched compositions or polymer synthons which are particularly well suited for incorporation during chemical assembly. When used to prepare modified PNA oligomers, applicants have observed unprecedented improvement in solubility and reduction in polymer self-aggregation. Consequently, for the first time known by applicants, it is possible to isolate purified PNA oligomers which do not adhere to the synthesis and sequence limitations well known in the art. Furthermore, using dot blot and Fluorescent In Situ Hybridization (PNA-FISH) formats (See for example: FIGS. 10A and 10B), applicants have demonstrated that PNAs which are modified with the preferred compositions of this invention exhibit hybridization properties which are not detectably different from the unmodified PNA oligomers. Thermal melting experiments have further confirmed that the preferred solubility enhancing moieties of this invention do not appreciably affect the Tm of polymer hybrids (See: Example 17). Interestingly however, further experimentation has demonstrated that the presence of the solubility enhancers can improve upon the cooperativity of the melting and reannealing transition without significantly affecting Tm (See: Example 18). In addition to the observed improvements in purity and ease of characterization, applicants have additionally observed improvements in polymer recovery when utilizing conventional chromatographic procedures for purification (See: Example 19). Taken as a whole, the data demonstrates that the PNAs which are modified with preferred modifying moieties exhibit no adverse affects on hybridization properties as compared with the unmodified polymers but are significantly more soluble in aqueous solution.
For the modification of peptides, polyamides and PNAS, suitably protected amino acids are typically used as the branched or multiply branched polymer synthons. One particularly useful synthon suitable for the modification of peptides, polyamides and PNAs, abbreviated herein as Fmoc-xe2x80x9cExe2x80x9daeg-OH, is the achiral, multiply branched, non-nucleophilic ether (compound 13) having the formula illustrated in FIG. 3A. Upon polymer modification, the modifying moiety is an ether moiety (herein referred to as xe2x80x9cExe2x80x9d) which is achiral, multiply branched, uncharged and non-nucleophilic.
Another particularly useful synthon suitable for modifying peptides, polyamides and PNAs, abbreviated herein as Fmoc-xe2x80x9c+xe2x80x9daeg-OH, is the achiral, multiply branched, non-nucleophilic zwitterion (compound 18) having the formula illustrated in FIG. 3B. Upon polymer modification, the modifying moiety is an ether moiety (herein referred to as xe2x80x9c+xe2x80x9d) which is achiral, multiply branched, non-nucleophilic and positively charged at physiological pH.
In another embodiment, this invention pertains to methods for improving the solubility of synthetic polymers such as nucleic acids, nucleic acid analogs, peptide polyamides and particularly PNA oligomers. The method comprises reacting the polymer, a monomeric subunit of a polymer or a synthesis support upon which a synthetic polymer is to be assembled, with one or more branched or multiply branched compositions or synthons useful for improving the solubility of synthetic polymers and/or which can minimize or eliminate polymer self-aggregation. Non-limiting examples of branched and multiply branched compositions suitable for the practice of the methods of this invention are described herein.
In still another embodiment, this invention relates to synthetic nucleic acids, nucleic acid analogs, polyamides, peptides, and particularly PNA oligomers, which have been modified with the compositions or methods described herein. Preferably, the synthetic polymers have been modified with simple branched or simple multiply branched compositions described herein.
In one preferred embodiment, the modified polymer comprises one or more achiral, multiply branched, non-nucleophilic, uncharged (neutral) ether modifying moieties (herein identified as xe2x80x9cExe2x80x9d) having the formula: 
In another preferred embodiment, the polymers of this invention comprise one or more achiral, multiply branched, non-nucleophilic, positively charged (at physiological pH) ether modifying moieties (herein identified as xe2x80x9c+xe2x80x9d) having the formula: 
The modified polymers of this invention may exist immobilized to supports including polymer arrays (e.g. the polymer may exist on the support on which it is assembled or may have been removed from the synthesis support, deprotected, purified, and re-immobilized to another support), as lyophilized powders or be dissolved or suspended in aqueous solution.
Moreover, this invention specifically relates to modified PNA oligomers and more preferably those modified PNA oligomers having a purine nucleobase content of 75% or greater in a PNA oligomer having 8 or more nucleobases. Likewise, this invention also relates to purified, unlabeled or labeled, modified PNA oligomers having four or more sequential G residues (nucleobases) in a PNA oligomer having 6 or more nucleobases. This invention additionally relates to purified, labeled or unlabeled, modified PNA oligomers having 6 or more sequential purine residues. As an extreme example of a purine-rich PNA oligomer, this invention relates to purified, labeled or unlabeled, homopurine modified PNA oligomers comprising 6 or more nucleobases.
In still another embodiment, the compositions of this invention-may also be offered in a kit or the methods used in combination with a kit. Preferred kits of this invention will comprise branched or multiply branched synthons so that one of ordinary skill in the art may easily utilize them during chemical assembly to thereby modify a synthetic polymer. Other preferred kits of this invention will comprise polymers which have been modified with one or more branched or multiply branched modifying moieties to thereby improve aqueous solubility of the polymer and/or decrease or eliminate polymer self-aggregation. Preferably, the kits comprise simple branched or simple multiply branched compositions described herein.
The kit-based compositions of this invention shall preferably be suitable for direct use in the chemical assembly of the polymer whether or not an automated instrument is utilized. Preferred kits of this invention will comprise Fmoc-xe2x80x9cExe2x80x9daeg-OH and/or Fmoc-xe2x80x9c+xe2x80x9daeg-OH. Alternatively, the kit shall comprise a synthesis support to which a composition (e.g. Fmoc-xe2x80x9cExe2x80x9daeg-OH and/or Fmoc-xe2x80x9c+xe2x80x9daeg-OH) or polymer comprising a modifying moiety has been covalently linked.
Consequently, when using the compositions, kits and/or methods described herein, it is now possible to routinely purify and characterize both labeled and unlabeled PNA oligomers having a purine content of 75% or greater. Additionally, it is now possible to routinely purify and characterize both labeled and unlabeled PNA oligomers having four or more sequential G residues. Furthermore, it is now possible to routinely purify and characterize labeled and unlabeled PNA oligomers having 6 or more sequential purine residues, including homopurine PNAs of at least 15 residues in length.
Guided by the teachings set forth herein, those of ordinary skill in the art will appreciate that the possession and/or practice of the embodiments of this invention will afford important features and advantages not presently known but which shall improve the state of the art. Several noteworthy features and advantages are summrarized as follows:
Unique Features and Advantages of the Methods, Kits and Compositions of this Invention
1. Improved aqueous solubility of polymers and particularly peptide nucleic acid polymers.
2. Minimize or eliminate polymer self-aggregation of polymers and particularly peptide nucleic acid polymers.
3. Facilitate synthesis, purification and analysis of many insoluble polymers and particularly purine-rich PNA polymers labeled with hydrophobic labels.
4. Produces little or no modification of the Tm of a hybrid between a modified PNA oligomer and a nucleic acid as compared with the hybrid formed with the unmodified PNA and a nucleic acid.
5. May improve the cooperativity of the melting and reannealing transitions of a hybrid formed from a modified PNA oligomer and a polymer or polymer segment comprising a complementary nucleobase sequence as compared with a hybrid wherein the PNA oligomer is unmodified.
6. Preferred PNA solubility enhancing compositions are achiral ethers and/or alcohols which comprise positively charged tertiary amines or uncharged moieties wherein the modifying moiety branches from the polymer backbone (e.g. a side chain).
7. Preferred PNA solubility enhancing compositions maintain subunit to subunit spacing which has been demonstrated to be the most favorable spacing so that the PNA exhibits optimal hybridization properties (i.e. comprise an N-[2-(aminoethyl)]glycine backbone).